© Joseph Sohm, Photo Researchers

When Michael Thomashow uprooted two decades ago from sunny southern California for his first faculty position in Pullman, Wash., he had trouble acclimating to the colder weather. That made him wonder how plants survive extreme temperature conditions. "Unlike us, they can't just get up and go inside," he muses. The Washington winters sowed the seeds of his interest in plant stress tolerance, and thus began Thomashow's pioneering work using Arabidopsis thaliana as a powerful model system. Since then, scientists have studied this humble little mustard plant for acidity, salinity, and other stresses.

THE WEED THAT CAME IN FROM THE COLD Before the mid-1980s, Arabidopsis researchers were growing their plants at a constant 20°C, says Gary Warren, University of London, UK. No one was stressing their precious model organisms. But after Thomashow broke from the pack and showed how easy it was to find cold-responsive genes (known as COR) in Arabidopsis, suddenly everyone wanted to do it.¹ "Stress-inducible genes were the new gold rush," says Warren. "With its

small genome, Arabidopsis looked like a rich seam."

Since then, Thomashow (now at Michigan State University) has discovered a family of cold-regulated transcriptional activators, called CBF proteins, that set in motion more than 30 COR genes. The COR proteins protect the cell from membrane damage and other cold-induced damage. The CBF factors are identical to DREB proteins that were independently discovered by Kazuo Shinozaki's group at the RIKEN Tsukuba Institute, Japan. Overexpression of one of these key transcription factors, CBF1, increases the freezing tolerance of even nonacclimated plants. A non-acclimated plant is one that hasn't had the chance to increase its freezing tolerance in response to decreasing temperatures. Normally, during cold acclimation, plants sense that the temperature is dropping and CBF1 naturally kicks in. For example, freezing kills nonacclimated rye at about -5°C, but acclimated rye can survive down to -30°C.

Tammy Irvine, Rear View Illustrations

This work has tremendous practical implications, says Thomashow: "If we knew more about the molecular response to these stresses, we might be able to develop novel ways to improve stress tolerance." To that end, Michigan State has filed patents for the key genes involved in the CBF/DREB1 pathway, and it has licensed the rights to Mendel Biotechnology, a Hayward, Calif.-based company that is currently testing the so-called Weatherguard technology. "We certainly know from lab tests that you can get an increase in freezing tolerance," says Thomashow, who is waiting to see if it will work in the field. "Technical success doesn't mean that you have a commercial product," cautions Mendel Biotechnology's Bill Goure. The company has sublicensed the technology so that it can be tested, and ultimately applied, over a broad range of plant types. If Weatherguard does eventually reach the market, "It will likely be the first technology coming out of plant functional genomics" says Goure.

Surviving the cold is not just about temperature. "The battery of genes affected by the CBF cold response also gives enhancement to drought tolerance, a finding first published by Shinozaki's group," says Thomashow. This makes sense, he says, since freezing damage is caused mostly by dehydration resulting from ice crystal formation. Not only do drought and freezing tolerance share common molecular response pathways, but they also go hand-in-hand as the top two agricultural problems worldwide. Goure suggests that Weatherguard's best asset may be its ability to increase drought tolerance. "In terms of what limits agriculture," says Thomashow, "drought is number one. Cold is number two." Number three is high salinity.

SALT SHAKER "Salt tolerance is a very serious agricultural problem and has been a major problem throughout human history," says Jian-Kang Zhu of the University of Arizona. In places with considerable rainfall, accumulated ions and salts are regularly washed out of the soil. But where rain is sparse, salts accumulate and eventually kill plant life. Zhu uses Arabidopsis as a model to study the SOS (salt overly sensitive) family of sodium-tolerant genes that he discovered, even though the glycophytic Arabidopsis is hardly salt-tolerant. Arabidopsis can acclimate to high-salt conditions the same way it can acclimate to plummeting temperatures, so it still has all the requisite molecular machinery for salt tolerance. By changing the expression of the SOS regulatory genes, Zhu explains, one can create a much more salt-tolerant plant, just as overexpressing CBF/DREB1 results in a more frost- tolerant plant.

Salt tolerance is studied in carnations by horticulturalists, in corn and wheat by agronomists, and in naturally salt-tolerant plants by biologists. "But the problem with these other plants is that the genetic infrastructure is not there, so you can't actually do a lot of functional studies," says Zhu. That's why Arabidopsis is the pick of the crop. Transformation of this plant, for example, "is really, really easy," says Zhu. Warren agrees: "If a company really wants to transform maize, it's possible. But it's not something you'd do in a routine lab. It's hell to get DNA into it."

The situation is changing somewhat. The salt-loving Thellungiella halophila, for example, may be the next best salt-stress model plant. Like Arabidopsis, it has a small genome, and more than 90% of its cDNAs have Arabidopsis counterparts.

But, says Zhu, "Arabidopsis is still the organism of choice." Friedrich Schöffl of the Eberhard-Karls-Universität Tübingen in Germany agrees. "The most important feature of any study system is genetics." He chose to study heat-shock responses in the mustard plant, even though it is not especially heat-tolerant compared to heartier botanic species such as tobacco. Thanks to its genetics, Arabidopsis beats the competition hands down.

ARABIDOPSIS AND ALUMINUM The tiny crucifer has also proven a heavy hitter in aluminum tolerance studies, says root biologist Leon Kochian of Cornell University. Aluminum toxicity is one of the hazards of acid soil, and it is a major agricultural problem because half of the world's potentially arable soil is acidic. Kochian puts it right up there with temperature, drought, and salt stress. Even though crop plants historically have been bred for aluminum tolerance, he says, there is ample room for improvement. Moreover, bioengineering is the only genetic solution for genetically homogenous plants that cannot be bred for aluminum tolerance.

Using Arabidopsis as a model system, Kochian and postdoc Owen Koekenga have recently identified two major quantitative trait loci involved in aluminum tolerance. They plan to overexpress aluminum tolerance genes in other plants, much the same way that the cold-response CBF/DREB1 genes are being overexpressed in crop plants to increase frost tolerance. When it comes to testing it in the field, Kochian stresses that genetics is only half the answer; reducing soil acidity is equally important.

Tammy Irvine, Rear View Illustrations

TIME TO FLOWER Not all environmental cues are stressful. Plants also regularly respond to daily or seasonal cues, such as diurnal light patterns and seasonal temperature changes. Wielding the scepter of Arabidopsis, scientists are making significant headway toward understanding the molecular machinery of these processes. For example, a slew of papers recently reported on the photo-period and flowering. George Coupland's group at the Max Planck Institute for Plant Breeding Research in Germany isolated a transcription factor, CO (constans) that activates a suite of target genes that regulate flowering time. They demonstrated that CO is expressed only during the interval of the day when plants are exposed to light on long days but are in darkness on short days. As day length gets longer, the plants start to flower.

Another piece in the molecular puzzle comes from Rick Amasino's lab at the University of Wisconsin-Madison. Amasino's group recently discovered another gene, ELF4 (early flowering 4), which is crucial for normal circadian rhythms.

Flowering time is also triggered by prolonged exposure to cold temperatures, which is different from the immediate cold-response that Thomashow studies. The longer wintry weather response, known as vernalization, can be simulated in the laboratory by refrigerating Arabidopsis for several weeks. By doing so, Amasino's group discovered FLC (flowering locus C), a key genetic player whose expression is turned off by winter and stays off long after the cold signal is gone. Since high levels of FLC prevent flowering, the plant is able to flower when spring arrives.

Caroline Dean and colleagues at the John Innes Center, Norwich, UK, study FRIGIDA, a flower-repressing gene that keeps FLC levels high before vernalization sets in. FRIGIDA ensures that "plants do not flower on a nice autumn day when the day-length is still relatively long." says Dean.

Like the plant-stress responses, seasonal responses potentially can be exploited for agricultural gain. "Expressing FLC would be important in crops like beets, spinach, and lettuce," says Amasino. "Flowering sort of destroys these crops. But it would take tremendously more resources in larger crop plants to do the kinds of things that people are doing with Arabidopsis. Now we have a baseline of information that can be applied to other systems. An important way for science to progress is to really understand one species and then do comparative work."

Leslie Pray (lpray@nasw.org) is a freelance writer in Northampton, Mass.